1. Field of the Invention
This invention relates to camptothecin compounds which inhibit the enzyme topoisomerase I and have anticancer activity. This invention is also related to the treatment of leukemia, solid tumors and brain glioma in mammals using the conjugates.
2. Background of the Invention
Camptothecin (CPT) is a naturally occurring cytotoxic alkaloid which is known to inhibit the enzyme topoisomerase I and is a potent anti-tumor agent. Camptothecin compounds have the general ring structure shown below. 
Camptothecin was first isolated from the wood and bark of Camptotheca acuminata by Wall et al. (Wall et al., 1966, J. Am. Chem. Soc., 88:3888).
Major synthetic efforts have been directed to derivatizing the A-ring and/or the B-ring attempting to improve cytotoxic activity and water-solubility.
U.S. Pat. No. 4,894,456 describes methods of synthesizing camptothecin compounds which act as inhibitors of topoisomerase I and are effective in the treatment of leukemia (L-1210). U.S. Pat. No. 5,225,404 discloses methods of treating colon tumors with camptothecin compounds. U.S. Pat. No. 4,894,456 describes methods of synthesizing camptothecin compounds which act as inhibitors of topoisomerase I and are effective in the treatment of leukemia (L-1210). U.S. Pat. No. 5,225,404 discloses methods of treating colon tumors with camptothecin compounds.
Numerous camptothecin compounds and their use as inhibitors of topoisomerase I are taught by U.S. Pat. No. 5,053,512; U.S. Pat. No. 4,981,968; U.S. Pat. No. 5,049,668; U.S. Pat. No. 5,106,742; U.S. Pat. No. 5,180,722; U.S. Pat. No. 5,244,903; U.S. Pat. No. 5,227,380; U.S. Pat. No. 5,122,606; U.S. Pat. No. 5,122,526; and U.S. Pat. No. 5,340,817. Analogs of camptothecin which show antiproliferative activity against human tumors both in vitro and in vivo have been developed and are described in U.S. Pat. Nos. 4,981,968; 5,049,668 and 5,122,526.
Brangi et al., Cancer Research, 59, 5938-5946 (1999), reports an investigation of camptothecin resistance in cancer cells and reports the compound difluoro-10,11-methylenedioxy-20(S)-camptothecin.
Of particular interest is the production of 10,11-methylenedioxy-20(S)-camptothecin (MDC) and analogs (U.S. Pat. No. 5,180,722) and alkylating analogs (U.S. Pat. No. 5,985,888). These MDC analogs have been shown to be highly active when tested against a number of human cancer cell lines (O""Connor, 1990 #2346; Adams et al.).
In order to increase the lifetime of the cleavable complexes with MDC, an analog was developed containing an alkylating chloromethyl group: 7-chloromethyl-10,11-methylenedioxy-20(S)-camptothecin, CMMDC (U.S. Pat. No. 5,559,235). This analog has been shown to alkylate the purine immediately 3xe2x80x2 to the cleavage site resulting in enhanced toxicity (Pommier, 1995 #2174; Valenti, 1997 #2344). The 7-bromomethyl-10,11-methylenedioxy-20(S)-camptothecin has also been described (U.S. Pat. No. 5,985,888) but probably due to the extreme reactivity of the bromomethyl group is not suitable for in vivo studies.
Glutathione (FIG. 2) is a tripeptide of glutamic acid, cysteine and glycine and found in high concentration in most normal cells and is often elevated in tumor tissue. In cancer cells, glutathione appears to play an important role in resistance to chemotherapy (Tew, 1994 #1038; Colvin, 1993 #628). For example, the chemotherapeutic alkylating agents such as cyclophosphamide, cisplatinum and BCNU are inactivated by covalent conjugation with glutathione to form thioether metabolites. This can occur spontaneously or through the action of the glutathione S-transferase enzymes. These transferases are often overexpressed in drug-resistant cells (Tew, 1994 #1038). Therefore glutathione conjugation results in chemical inactivation of the drug as well as increased export of the drug from the cell leading to resistance.
Peptide derivatives of camptothecin have been made. For example, a number of derivatives created by formation of amide linkages between the carboxylate of amino acids and peptides and the amine of 9-amino-camptothecins have been proposed to improve solubility (U.S. Pat. No. 5,180,722). A derivative containing a xcex3-glutamyl-linkage to the 9-amino position has been patented as a prodrug to deliver camptothecin to cells which express xcex3-glutamyltranspeptidase (U.S. Pat. No. 5,854,006). Peptide derivatives have also been prepared as linker arms between camptothecin and polymers to improve drug delivery (U.S. Pat. No. 5,892,043).
Formation of glutathione conjugates of anticancer drugs has been reported in some instances to result in inactivation or increased transport of the drugs out of the cell. There are however several examples of glutathione conjugates which appear to bind to and inhibit enzymes and therefore be potentially useful therapeutic agents. The glutathione conjugate of the anticancer drugs doxorubicin and daunorubicin (Gaudiano et al, JACS 1994, 116, 6537; claimed in U.S. Pat. No. 5,646,177) inhibit some of the drug efflux proteins and used in combination with other agents may be beneficial therapeutic agents (Asakura, 1997 #1749; Priebe, 1998 #2179).
Another anticancer agent has been designed as a glutathione conjugate analog. This agent, the peptidomimetic xcex3-glutamyl-S-(benzyl)cysteinyl-R(xe2x88x92)-phenyl-glycine (TER 117) is designed to inhibit glutathione 5-transferase. In vivo activity against cancer cell lines is realized when the diethyl ester derivative (TER199) was prepared (Morgan, 1996 #2348).
A glutathione conjugate has also been shown to bind to a DNA-dependent protein kinase (Shen, 1999 #2349). This kinase can bind DNA (structural similarities to topoI?)
A need continues to exist, however, for camptothecin compounds having improved biological activity. The present invention is directed to camptothecin compound which may be prepared by the reaction of a cysteine containing group with an alkylating camptothecin.
Accordingly, one object of the present invention is to provide novel camptothecin compounds.
Another object of the present invention is to provide a method of treating leukemia or solid tumors in a mammal in need thereof by administration of a camptothecin-peptide conjugate.
Another object of the present invention is to provide a method of inhibiting the enzyme topoisomerase I and/or alkylating DNA of associated DNA-topoisomerase I by contacting a DNA-topoisomerase I complex with a camptothecin-peptide conjugate.
Another object of the present invention is to provide a method of stabilizing the enzyme topoisomerase I and/or alkylating DNA of associated DNA-topoisomerase I by contacting a DNA-topoisomerase I complex with a camptothecin-peptide conjugate.
These and other objects are made possible by the following camptothecin compounds which have combined topoisomerase I inhibiting and DNA-topoisomerase I cleavable complex stabilizing properties, of the formula 
wherein R1 and R2, are each independently
NO2, NH2, H, F, Cl, Br, I, COOH, OH, Oxe2x80x94C1-6 alkyl, SH, Sxe2x80x94C1-6 alkyl, CN, NHxe2x80x94C1-6 alkyl, N(C1-6 alkyl)2, CHO, C1-8 alkyl, N3,
xe2x80x94Zxe2x80x94(CH2)axe2x80x94Nxe2x80x94((CH2)bOH)2, wherein Z is selected from the group consisting of O, NH and S, and a and b are each independently an integer of 2 or 3,
xe2x80x94Zxe2x80x94(CH2)axe2x80x94Nxe2x80x94(C1-6 alkyl)2 wherein Z is selected from the group consisting of O, NH and S, and a is an integer of 2 or 3,
xe2x80x94CH2NR4R5 where (a) R4 and R5 are, independently, hydrogen, C1-6 alkyl, C3-7 cycloalkyl, C3-7 cycloalkyl-C1-6 alkyl, C2-6 alkenyl, hydroxy-C1-6 alkyl, C1-6 alkoxy-C1-6 COR6 where R6 is hydrogen, C1-6 alkyl, perhalo-C1-6 alkyl, C3-7 cycloalkyl, C3-7 cycloalkyl-C1-6 alkyl, C2-6 alkenyl, hydroxy-C1-6 alkyl, C1-6 alkoxy, C1-6 alkoxy-C1-6 alkyl, or (b) R4 and R5 taken together with the nitrogen atom to which they are attached form a saturated 3-7 membered heterocyclic ring which may contain a O, S or NR7 group, where R7 is hydrogen, C1-6 alkyl, perhalo-C1-6 alkyl, aryl, aryl substituted with one or more groups selected from the group consisting of C1-6 alkyl, halogen, nitro, amino, C1-6 alkylamino, perhalo-C1-6 alkyl, hydroxy-C1-6 alkyl, C1-6 alkoxy, C1-6 alkoxy-C1-6 alkyl and xe2x80x94COR8 where R8 is hydrogen, C1-6 alkyl perhalo-C1-6 alkyl, C1-6 alkoxy, aryl, and aryl substituted with one or more C1-6 alkyl, perhalo-C1-6 alkyl, hydroxy-C1-6 alkyl, or C1-6 alkoxy-C1-6 alkyl groups;
R3 is H; or
or R2 and R3 combine to form a ring 
where R9 and R10 are each independently H or F and n is an integer of 1 or 2;
R11 is H, or C(O)xe2x80x94(CH2)mxe2x80x94NR12R13, where m is an integer of 1-6 or xe2x80x94C(O)CHR14NR12R13, where R14 is the side chain of one of the naturally occurring xcex1-amino acids, R12 and R13 are, independently, hydrogen, C1-8 alkyl or xe2x80x94C(O)CHR15NR16R17, where R15 is the side chain of one of the naturally occurring xcex1-amino acids and R16 and R17 are each independently hydrogen or C1-8 alkyl;
R18 is OR19 or R19OC(O)xe2x80x94(CH2)mxe2x80x94NR20, or R21OC(O)CHR22NR20, where R19 is H or C1-6 alkyl, m is an integer of 1-6, R22 is the side chain of one of the naturally occurring xcex1-amino acids, R20 is hydrogen, C1-8 alkyl or xe2x80x94C(O)CHR23NR24R25, where R23 is the side chain of one of the naturally occurring xcex1-amino acids and R24 and R25 are each independently hydrogen or C1-8 alkyl;
R26 is H or 
where R27 is H or C1-6 alkyl; and
X is S or O,
or a pharmaceutically acceptable salt thereof.